Evaluation of Resistance Induction Promoted by Bioactive Compounds of Pseudomonas aeruginosa LV Strain against Asian Soybean Rust

Pseudomonas are known as higher producers of secondary metabolites with antimicrobial properties and plant growth promoters, including resistance induction. These mechanisms should be an alternative to pesticide use in crop production. Phakopsora pachyrhizi causes Asian soybean rust, representing a high loss of yield around the world. The objective of this paper was to evaluate the application of secondary metabolites produced by Pseudomonas aeruginosa LV strain from the semi-purified fraction F4A in soybean plants to induce plant resistance against P. pachyrhizi in field conditions. The experimental design was performed in randomized blocks with three replicates using two F4A doses (1 and 10 μg mL−1) combined or not with fungicides (Unizeb Gold® or Sphere Max®). The control treatment, with Uni + Sph, saponins, flavonoids, and sphingolipids, showed higher intensities in the plants. In contrast, plants treated with the F4A fraction mainly exhibited fatty acid derivatives and some non-identified compounds with nitrogen. Plants treated with Sphere Max®, with or without F4A10, showed higher intensities of glycosylated flavonoids, such as kaempferol, luteolin, narigenin, and apigenin. Plants treated with F4A showed higher intensities of genistein and fatty acid derivatives. These increases in flavonoid compound biosynthesis and antioxidant properties probably contribute to the protection against reactive oxygen species (ROS).


Introduction
In the 2022/23 season, 78.5 million hectares were cultivated with soybean, and the yield was 322.8 million tons.Brazil is the one of biggest soybean producers and the fifthlargest nation in agricultural land area.In Brazil, the most widely grown crop is soybean (Glycine max L.), with 44 million ha sown and a yield of 154.6 million tons, followed by corn (Zea mays L.), with 22.6 million ha sown and a yield of 131.8 million tons in the 2022/23 season [1].After thousands of years of use as an essential source of feed for humans and animals, soybean has emerged as one of the most significant crops on the global market and is the most important protein source for animal and human food [2].
There are some fungi causing soybean disease, such as anthracnose, stem canker, leaf blight, purple seed, downy mildew, powdery mildew, damping-off, and stem rot, and infections are caused by bacteria, viruses, and nematodes.However, none of them can match the destructive potential and economic impact of Asian soybean rust (ASR), caused by P. pachyrhizi.ASR should potentially yield losses of up to 90%, which corresponds to a billion-dollar loss on a global scale [3].
Urediniospore germination initiates the ASR infection process, which is subsequently followed by the development of germ tubes and appressoria [4,5].The appressoria infect epidermal cells, and hypha grow through the intercellular spaces.The formation of haustoria in mesophyll cells, visible eruptions on the epidermis, and uredia occur around after 12 days, causing chlorosis and premature defoliation [6,7].
Some agricultural practices such as crop rotation, early-maturing cultivars, eliminating alternative plant hosts, and implementing sanitary voids are effective measures to mitigate the impacts of ASR.However, chemical control remains the most common and effective method to control P. pachyrhizi, preventing the secondary cycle of ASR and ensuring high productivity [8,9].
The Food and Agriculture Organization (FAO) [10] reported that in the year 2020, a significant amount of 2.7 million tons (Mt) of fungicide were used worldwide, resulting in the production of 7.2 Mt of formulated pesticide products valued at around USD 41.1 billion.The amount of fungicide used has increased 50% when compared with the levels in the 1990s.Brazil is the second largest consumer of pesticides, with a consumption of 377 kilotons (kt), closely behind the USA, which leads the use charts with 408 kt.
However, it is crucial to acknowledge that the use of pesticides carries potential risks of environmental contamination and for human and animal health.In addition, the exposure to pesticides can increase the emergence of pathogen-resistant microorganisms [11,12].Fungal infections have been effectively controlled by site-specific fungicides like demethylation inhibitors (DMIs) and quinone outside inhibitors (QoIs) for years, but their efficiency has been decreasing [13,14].
The challenge in agricultural research involves the identification of novel molecules, specifically bioactive compounds with antifungal activity, as these compounds show low environmental impact and low risks for human and animal health.In the last decade, innovative strategies for pest and disease management have emerged, emphasizing the use of compounds that trigger natural defense mechanisms in plants.These methods focus on mechanisms such as Inducing Systemic Resistance (ISR) or Systemic Acquired Resistance (SAR), which seek to improve plant defense response against pest and plant pathogens [16][17][18][19][20].
The Pseudomonas genera is found in the whole of the environment around the world.Some species are described as biocontrol agents, producing compounds such as phenazines, indolinones, peptides, glycopeptides, lipids, and aliphatic compounds with antimicrobial activity against fungi and bacteria pathogens for plants and humans [21][22][23][24].
The objective of this paper was to evaluate the effect of the semi-purified fraction F4A produced by P. aeruginosa LV strain on SAR activation in soybeans infected with P. pachyrhizi by metabolomic analysis.

Production and Extract of Microbial Bioactive Compounds
P. aeruginosa strain LV was initially isolated from an old citrus canker lesion (Citrus sinensis cv.Valencia) in an orange leaf and is maintained in the collection of the Microbial Ecology Laboratory, Londrina, Brazil.
To produce bioactive compounds, P. aeruginosa LV strain was cultured in nutrient broth plus 5 mg L −1 CuCl 2 for 10 days at 28 • C.After that, the culture was centrifuged at 9000 rpm for 15 min at 4 • C (Sorval RC-5C, Thermo Fisher Scientific, Waltham, MA, USA) and the supernatant was kept and the pellet discarded.The supernatant was reduced to 10% of its original volume in a chamber at 60 • C (SS Scientific, Londrina, Brazil).This concentrated sample was partitioned with dichloromethane (2:1, v/v) three times to obtain the dichloromethane phase (DP).DP was once concentrated with a rotatory evaporator (Büchi R-215, BUCHI Labortechnik, Flawil, Switzerland) at 45 • C at 100 rpm −1 , and subsequently, it was submitted for vacuum liquid chromatography in a glass column (20 mm de diameter, 350 mm length) with silica gel 60 (0.063-0.200 mm, Merck, Darmstadt, Germany) as the stationary phase.The mobile phase applied (400 mL) was dichloromethane and ethyl acetate 1:1 (v/v) to obtain the fraction F4A [30].

Effect of F4A Fraction on Asian Soybean Rust
The experimental design was randomized from six treatments and three replicates and evaluated two concentrations of F4A (1 and 10 µg mL −1 ) added or not to Unizeb Gold ® (Uni) (UPL, Ituverava, Brazil) and/or Sphere Max ® (Sph) (Bayer S.A., São Paulo, Brazil), according to the manufacturers' instructions, to evaluated the following treatments: F4A1, F4A1 + Uni, F4A1 + Sph, F4A10, F4A10 + Uni, and F4A10 + Sph.The positive control (Unizeb Gold ® and Sphere Max ® ) was an additional group named Uni + Sph.The parcel had a total area of 9 m 2 and ten lines.
The F4A fraction and pesticides were applied with a backpack sprayer with a CO 2 cylinder (Agro Pesquisa, Campinas, Brazil) with a flow of 200 L ha −1 .The application was carried out two times (A1 and A2).A1 was carried out in R5.1, right after the first symptoms of Asian soybean rust, and A2 was carried out in R6.After 24 h for each application, five leaves were collected from each parcel and frozen in dry ice.

Metabolomic Analysis 2.3.1. Sample Preparation
From the five leaves collected in the field, the healthy leaves were selected, washed, and powdered by liquid nitrogen in a pistil.From these materials, 0.2 g was extracted with 10 mL methanol and water 8:2 (v/v) in an ultrasonic bath (30 min) (Branson 1510-DTH, Marshall Scientific, Hampton, NH, USA); subsequently, the samples were centrifuged for 10 min at 6.000 rpm and 8 • C (Jouan CR3i, Thermo Fisher Scientific, Waltham, MA, USA).The supernatants were filtered by Millex syringe filters (PTFE, 0.22 mm × 13 mm, Merck Millipore, Burlington, MA, USA) and added to vials of 1.5 mL to carry out the chromatography analysis.Aliquots of 20 µL of each sample were pooled to prepare the quality control sample (QC).

Metabolomic Analysis
The metabolomic data analysis was carried out in an Ultra-Fast Liquid Chromatograph LC-20AD Shimadzu Prominence (Shimadzu, Kyoto, Japan) coupled to a diode array detector and mass spectrophotometer with ionization source electrospray and analyzer quadrupole and time of flight (MicrOTOF-Q III Bruker Daltonics, Billerica, MA, USA).
The samples (1 µL) were processed in the chromatographic system using a Kinetex C18 column (2.6 µm, 100 A, 150 × 2.1 mm, Phenomenex, Torrance, CA, USA).The mobile phase was composed of acetonitrile (B) and water (A), with formic acid 0.1% (v/v) added to both phases.The gradient elution profile was the following: 0-2 min 3% B, 2-25 min 3 to 25% B, 25-40 min 25 to 80% B, and 40-43 min 80% B. The flow rate was 0.3 mL min −1 , and the chromatographic column was maintained at 50 • C during the analysis.Samples were analyzed in positive and negative ion mode.Nitrogen was used as a nebulizer (4 Bar) and drier (9 L/min) and collision gas.The compound annotation was based on the spectral data from UV, MS, and the fragmentation profile compared with the databases and data reported in the literature [35][36][37].Some compound data were confirmed by the co-injection of authentic standards.
The data were initially analyzed by Data Analysis 4.2 software (Bruker Daltomics, Billerica, MA, USA)) and were subsequently aligned and reduced by the software Metalign (version 011012) and MSClust (version 300817), respectively.The statistical analyses were performed by the platform Metaboanalyst 5.0, and the data were previously normalized by median, log transformed, and auto-scaled before these analyses.The statistical analyses include hierarchical cluster heatmap (HCH) and principal component analysis (PCA).

Results
The dataset was composed of 129 entrances after data processing.The spectral data and annotated compounds from selected entrances obtained by statistical analyses are summarized in Table 1.
Figure 1 presents the principal component analysis (PCA) score plot from the LC-MS data of soybean leaf samples infected with Phakopsora pachyrhizi under different treatments.PCA is an unsupervised statistical technique that transforms high-dimensional data into a lower-dimensional coordinate system, preserving the highest variance in the data.In the PCA analysis, group 1, represented by red triangles, includes the treatments Uni + Sph F4A1 + Sph, F4A10 + Sph, and F4A10, while group 2, represented by green plus symbols, comprises the treatments F4A1, F4A1 + Uni, and F4A10 + Uni.The two groups show a tendency of separation between the groups (Figure 1).
Figure 2 shows a heatmap and hierarchical clustering analysis (HCA) of soybean leaf samples infected with P. pachyrhizi under different treatments.The heatmap visualizes the relative intensity of specific compounds in the samples, where darker red colors indicate higher intensity.The HCA groups the samples based on the similarities in their metabolic profiles, highlighting four main clusters: A, B, C, and D.

Discussion
Unizeb Gold ® is a multi-site fungicide.Its active ingredient is mancozeb, which belongs to the group of dithiocarbamates.When exposed to water, mancozeb breaks down and releases ethylene bisisothiocyanate sulfide (EBIS).This compound is then converted into ethylene bisisothiocyanate (EBI) by UV light.The mode of action is not fully elucidated; however, EBIS and EBI are believed to be toxic to enzymes containing sulfhydryl groups [39].
Those effects were observed in a field experiment on soybeans [45].The treatments Uni + Sph, F4A1 + Sph, and F4A10 + Sph were highly effective in controlling ASR, delaying disease progression, and causing damage to the spores and hyphae of P. pachyrhizi.In the same study, Barazetti et al. [45] observed in an in vitro experiment that F4A1 and F4A10 inhibit spore germination and germinative tube growth.
Soybean treated with Sphere Max ® increased production of flavonoids, which suggested that Sphere Max ® induces plant defense.Strobilurins have been related as inducing priming responses in Arabidopsis [46], and the same results were observed in Nicotiana tabacum treated with pyraclostrobin, decreasing tobacco mosaic virus [47], and in cucumber treated with the same compound against mosaic virus and P. syringae pv.tomato [48].
Despite the resistance inducing effects, there is no data in the literature that associate the application of the active ingredients present in Unizeb Gold ® and Sphere Max ® with the biosynthesis of flavonoids as observed in our investigation.
Plants treated with Uni + Sph also exhibit higher levels of saponins.This one is part of the terpene class, mainly present in dicotyledonous species, and is related to insect and fungal attack in plants.In both cases, this activity is commonly linked to their interaction with biological membranes, forming complexes with sterols, proteins, or phospholipids.More recently, it was verified that saponins also have the potential to activate defense responses through salicylic acid induction and oxidative burst in Brassica napus against Leptosphaeria maculans [49].Sadly, the interaction between saponin levels and P. pachyrhizi infection on soy plants is still unclear.
The F4A fraction obtained from the P. aeruginosa LV strain contains two phenazines, compounds that have been reported as biocontrol agents through antagonism to other microorganisms in the rhizosphere besides being important to control fungal crop diseases [50].In general, phenazines, especially pyocyanin, promote the generation of ROS, which act as signaling molecules involved in growth processes, development, and defense responses against pathogens through ISR [51].
The inoculation of P. aeruginosa in rice stimulated plant defense against Magneporthe grisea, which causes blast disease, and decreased infection.Pyocyanin (PYO) was crucial as an elicitor of ISR.When rice was treated with 25 ηM, 1 ηM, and 100 ηM of PYO, the infection of M. grisea was reduced, but no effect was observed with Rhizoctonia solani [52].The ISR should explain the effect observed on P. pachyrhizi in our experiment, where soybeans treated with 1 µg F4A increased soybean yield to 4.64 ton ha −1 , 6.2% more than plants treated with Uni + Sph.Another study demonstrated that F4A activates soybean defenses, increasing the expression of phenylalanine ammonia lyase (PAL), Omethyltransferase (OMT), and pathogenesis-related protein-2 (PR-2; glucanases) defenserelated genes, detected 24 and 72 h after soybean sprouts were sprayed with purified Fluopsin C, which is one of the compounds that contains the F4A fraction [53].
The enzyme PAL converts phenylalanine to trans-cinnamic acid and ammonia; the first one should be incorporated into many phenolic compounds and is present in esters, coumarins, lignin, and flavonoid formation [54].It is largely known that flavonoids have antioxidant properties due to the presence of B-ring catechol groups and other factors which donate electron-reducing ROS [55].The production of phytoalexin glyceollin is one of the most common responses of soybeans against phytopathogenic fungi.Silva et al. [54], studying soybean leaves inoculated and not inoculated with P. pachyrhizi, observed that this phytoalexin was only expressed in the second group.The metabolic precursor of glyceollin is daidzein, which is present in soybean plants treated with F4A.Both glyceollin and daidzein have antioxidant activity against ROS [56][57][58], as well as other compounds of the flavonoid group that were identified in plants treated with F4A, such as kaempferol [59,60], apigenin [61], and luteolin [55], and should be related to the plant protection against P. pachyrhizi observed in our results.Gupta et al. [62], studying Arabidopsis thaliana plants treated with ethylene, a hormone known to be involved in stress responses, observed that there was an increase in isoflavonoid accumulation when compared to control plants, especially genistin (2.7 fold), daidzein (21.38 fold), and genistein (7.6 fold).They also verified an increment in levels of fatty acids and flavonoids, suggesting an important role of these compounds in plant defense besides being part of the ethylene pathway.
Plants treated with F4A10 + Sph increased sphingolipid and pheophorbide synthesis.Sphingolipids are important for cell membrane structure, cell-to-cell signalization, cell wall formation, stomatal closure, and controlled cell death [63,64].Pheophorbide A is derived from chlorophyl degradation during the natural process of leaf senescence after seed formation [65] or by biotic and abiotic stress or fungal infection as in P. pachyrhizi [7].
In general, all treatments showed the presence of fatty acids or their derivates, such as octadecatrienoic acid and eicosatetraenoic acid, which were probably related to the grain formation of soybeans during our first (R5) and second (R6) harvests.Once stored, seeds that have these lipids as their main reserve form are subjected to slow and consistent exposure to oxygen, forming hydroperoxides, other oxygenated acids, and free radicals [66].
Lipids and fatty acids can also act in defense responses to biotic and abiotic stresses.Linolenic and linoleic acids, for example, are precursors of oxylipins in plants, a product of self-oxidation or enzymatic oxidation [54].
Through the heatmaps, it is also possible to observe some variations in the production of compounds, even within the same treatments.This probably stems from the fact that the experiment was conducted under field conditions, where a series of biotic and abiotic factors, including temperature, humidity, wind, pests, and weeds, cannot be fully controlled [67].
In conclusion, our results suggest that the F4A fraction induced plant resistance against Asian soybean rust, decreasing lesion formation and increasing soybean yield.

Figure 3 .
Figure 3. Heatmap of the soybean leaf samples infected with Phakopsora pachyrhizi from control (red) and the fraction F4A1 (green) treatments.

Figure 3 .
Figure 3. Heatmap of the soybean leaf samples infected with Phakopsora pachyrhizi from control (red) and the fraction F4A1 (green) treatments.

Table 1 .
Annotated compounds from samples of controls and treatments with F4A.